Free radicals have been shown to inhibit tumor growth by causing oxidative damage to lipids, proteins, and nucleic acids of the tumor cells. In clinical practice, a photo-sensitizer is first delivered to a tumor site and then activated by irradiation to generate free radicals, thus inhibiting tumor growth. Among known photo-sensitizers, Photofrin II has recently been approved by the U.S. Food and Drug Administration. Preparation of Photofrin II is tedious.
Fullerenes are conjugated olefins of a closed cage structure. When photo-excited, they are capable of transforming molecular oxygen into singlet oxygen and then the related free radicals, such as superoxide free radicals, i.e., O2xe2x88x92. However, fullerenes have low bioavailability and must be chemically modified before they can be tested for their efficacy, if any, as photo-sensitizers in treating tumor.
One aspect of this invention relates to multioligoanilinated fullerenes (MOAFs) of the following formula: 
In this formula, p and each q, independently, is an integer of 0-20; each a is an integer of 1-8; each b is 0 or 1; each c is an integer of 1-20, provided that when b is 0, c is 1; each n is 1 or 2; m is an integer of 1-20; F1 and each F2, independently, is a C60-66 or C70-76 fullerene, preferably a C60-66 or C70 fullerene; each of S and T, independently, is xe2x80x94OH, xe2x80x94NH2, xe2x80x94NHR, or xe2x80x94SH, wherein R is C1-20 alkyl; each A, independently, is an oligoaniline, the term xe2x80x9coligoanilinexe2x80x9d referring to a linear chemical species, the backbone of which consists of 2-12 aniline units; each nitrogen atom of the oligoaniline is optionally substituted with xe2x80x94Z, xe2x80x94CH2xe2x80x94COxe2x80x94OH, xe2x80x94CH2xe2x80x94COxe2x80x94Oxe2x80x94Z, xe2x80x94CH2xe2x80x94COxe2x80x94Sxe2x80x94Z, xe2x80x94CH2xe2x80x94COxe2x80x94NH2, or xe2x80x94CH2xe2x80x94COxe2x80x94NHxe2x80x94Z; and each benzene ring is optionally substituted with xe2x80x94Oxe2x80x94Z, xe2x80x94Sxe2x80x94Z, xe2x80x94NHxe2x80x94Z; Z being xe2x80x94Exe2x80x94D, wherein E is xe2x80x94Rxe2x80x94, xe2x80x94Rxe2x80x94Arxe2x80x94, xe2x80x94Arxe2x80x94Rxe2x80x94, or xe2x80x94Arxe2x80x94; and D is xe2x80x94OH, xe2x80x94SH, xe2x80x94NH2, xe2x80x94NHOH, xe2x80x94SO3H, xe2x80x94OSO3H, xe2x80x94CO2H, xe2x80x94CONH2, xe2x80x94CHxe2x80x94(NH2)xe2x80x94CO2H, P(OH)3, xe2x80x94PO(OH)2, xe2x80x94Oxe2x80x94PO(OH)2, xe2x80x94Oxe2x80x94PO(OH)xe2x80x94Oxe2x80x94PO(OH)2, xe2x80x94Oxe2x80x94PO(Oxe2x88x92)xe2x80x94Oxe2x80x94CH2CH2NH3+, -glycoside, xe2x80x94OCH3, xe2x80x94OCH2(CHOH)4xe2x80x94CH2OH, xe2x80x94OCH2(CHOH)2xe2x80x94CH2OH, xe2x80x94C6H3(OH)2, xe2x80x94NH3+, xe2x80x94N+H2Rb, xe2x80x94N+HRbRc, or xe2x80x94N+RbRcRd, each of Rb, Rc, and Rd, independently, being C1-20 alkyl; and Ar being aryl; each K, independently, is xe2x80x94H, xe2x80x94[N(X)xe2x80x94C6H4]1-3xe2x80x94NH2, xe2x80x94[N(X)xe2x80x94C6H4]1-3xe2x80x94NHxe2x80x94C(xe2x95x90S)xe2x80x94SH, xe2x80x94[N(X)xe2x80x94C6H4]1-3xe2x80x94Nxe2x95x90CHxe2x80x94Arxe2x80x94SH, xe2x80x94[N(X)xe2x80x94C6H4]1-3xe2x80x94NHxe2x80x94COxe2x80x94Arxe2x80x94SH, wherein X is xe2x80x94H, xe2x80x94Z, xe2x80x94CH2xe2x80x94COxe2x80x94OH, xe2x80x94CH2xe2x80x94COxe2x80x94Oxe2x80x94Z, xe2x80x94CH2xe2x80x94COxe2x80x94Sxe2x80x94Z, xe2x80x94CH2xe2x80x94COxe2x80x94NH2, xe2x80x94CH2xe2x80x94COxe2x80x94NHxe2x80x94Z; and Ar is aryl; each G, independently, is xe2x80x94Oxe2x80x94Bxe2x80x94Rxe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94Bxe2x80x94Rxe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94Bxe2x80x94Rxe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94Bxe2x80x94Rxe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94Bxe2x80x94Rxe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94Bxe2x80x94Rxe2x80x94Sxe2x80x94, wherein R is C1-30 alkyl; B, independently, is xe2x80x94R1xe2x80x94Oxe2x80x94[Si(CH3)2xe2x80x94Oxe2x80x94]1-1000, C1-2000 alkyl, C6-40 aryl, C7-60 alkylaryl, C7-60 arylalkyl, (C1-30 alkyl ether)1-100, (C6-40 aryl ether)1-100, (C7-60 alkylaryl ether)1-100, (C7-60 arylalkyl ether)1-100, (C1-30 alkyl thioether)1-100, (C6-40 aryl thioether)1-100, (C7-60 alkylaryl thioether)1-100, (C7-60 arylalkyl thioether)1-100, (C2-50 alkyl ester)1-100, (C7-60 aryl ester,1-100, (C8-70 alkylaryl ester)1-100, (C8-70 arylalkyl ester)1-100, xe2x80x94R1xe2x80x94COxe2x80x94Oxe2x80x94(C1-30 alkyl ether)1-100, xe2x80x94R1xe2x80x94COxe2x80x94Oxe2x80x94(C6-40 aryl ether)1-100, xe2x80x94R1xe2x80x94COxe2x80x94Oxe2x80x94(C7-60 alkylaryl ether)1-100, xe2x80x94R1xe2x80x94COxe2x80x94Oxe2x80x94(C7-60 arylalkyl ether)1-100, (C4-50 alkyl urethane)1-100, (C14-60 aryl urethane)1-100, (C10-80 alkylaryl urethane1-100, (C10-80 arylalkyl urethane)1-100, (C5-50 alkyl urea)1-100, (C14-60 aryl urea)1-100, (C10-80 alkylaryl urea)1-100, (C10-80 arylalkyl urea)1-100, (C2-50 alkyl amide)1-100, (C7-60 aryl amide)1-100, (C8-70 alkylaryl amide)1-100, (C8-70 arylalkyl amide)1-100, (C3-30 alkyl anhydride)1-100, (C8-50 aryl anhydride)1-100, (C9-60 alkylaryl anhydride)1-100, (C9-60 arylalkyl anhydride)1-100, (C2-30 alkyl carbonate)1-100, (C7-50 aryl carbonate)1-100, (C8-60 alkylaryl carbonate)1-100, (C8-60 arylalkyl carbonate)1-100, xe2x80x94R1xe2x80x94Oxe2x80x94COxe2x80x94NHxe2x80x94(R2 or Arxe2x80x94R2xe2x80x94Ar)xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94(C1-30 alkyl ether, C6-40 aryl ether, C7-60 alkylaryl ether, or C7-60 arylalkyl ether)1-100, xe2x80x94R1xe2x80x94Oxe2x80x94COxe2x80x94NHxe2x80x94(R2 or Arxe2x80x94R2xe2x80x94Ar)xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94(C2-50 alkyl ester, C7-60 aryl ester, C8-70 alkylaryl ester, or C870 arylalkyl ester)1-100, xe2x80x94R1xe2x80x94Oxe2x80x94COxe2x80x94NHxe2x80x94(R2 or Arxe2x80x94R2xe2x80x94Ar)xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94(C1-30 alkyl ether, C6-40 aryl ether, C7-60 alkylaryl ether, or C7-60 arylalkyl ether)1-100xe2x80x94CO-NH-(R2 or Arxe2x80x94R2xe2x80x94Ar)xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94, xe2x80x94R1xe2x80x94Oxe2x80x94COxe2x80x94NHxe2x80x94(R2 or Arxe2x80x94R2xe2x80x94Ar)xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94(C2-50 alkyl ester, C7-60 arylaryl ester, C8-70 alkylaryl ester, or C8-70 arylalkyl ester)1-100xe2x80x94R3xe2x80x94Oxe2x80x94COxe2x80x94NHxe2x80x94(R2 or Arxe2x80x94R2xe2x80x94Ar)xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94, xe2x80x94R1xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94(R2 or Arxe2x80x94R2xe2x80x94Ar)xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94(C1-30 alkyl ether, C6-40 aryl ether, C7-60 alkylaryl ether, or C7-60 arylalkyl ether)1-100, xe2x80x94R1xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94(R2 or Arxe2x80x94R2xe2x80x94Ar)xe2x80x94NHxe2x80x94COxe2x80x94(C2-50 alkyl ester, C7-60 aryl ester, C8-70 alkylaryl ester, or C8-70 arylalkyl ester)1-100, xe2x80x94R1xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94(R2 or Arxe2x80x94R2xe2x80x94Ar)xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94(C1-30 alkyl ether, C6-40 aryl ether, C7-60 alkylaryl ether, or C7-60 arylalkyl ether)1-100xe2x80x94COxe2x80x94NHxe2x80x94(R2 or Arxe2x80x94R2xe2x80x94Ar)xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94, xe2x80x94R1xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94(R2 or Arxe2x80x94R2xe2x80x94Ar)xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94(C2-50 alkyl ester, C7-60 aryl ester, C8-70 alkylaryl ester, or C8-70 arylalkyl ester)1-100xe2x80x94R3Oxe2x80x94COxe2x80x94NHxe2x80x94(R2 or Arxe2x80x94R2xe2x80x94Ar)xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94, xe2x80x94R1xe2x80x94Oxe2x80x94COxe2x80x94NHxe2x80x94(R2 or Arxe2x80x94R2xe2x80x94Ar)xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94(C2-50 alkyl amide, C7-60 aryl amide, C8-70 alkylaryl amide, or C8-70 arylalkyl amide)1-100, or xe2x80x94R1xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94(R2 or Arxe2x80x94R2xe2x80x94Ar)xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94(C2-50 alkyl amide, C7-60 aryl amide, C8-70 alkylaryl amide, or C8-70 arylalkyl amide)1-100; wherein each of R1, R2, and R3, independently, is C1-30 alkyl; and Ar is aryl.
Also within the scope of this invention are pharmaceutically acceptable salts of the MOAFs described above. Such a salt can be formed between a negatively charged ionic group (e.g., sulfonate or carbonate) in an MOAF and a positively charged counterion (e.g., a sodium ion). Likewise, a positively charged ionic group (e.g., ammonium) in an MOAF can also form a salt with a negatively charged counterion (e.g., chloride).
One subset of the MOAFs of this invention are featured by that a is an integer of 3-6. Another subset of the MOAFs are featured by that b is 1, or b is 0 and c is 1. Still another subset of the MOAFs are featured by that n is 2. Yet stilled another subset of the MOAFs are featured by that A is tetraaniline, optionally substituted at nitrogen atoms with Z; E is xe2x80x94Rxe2x80x94 or xe2x80x94Rxe2x80x94Arxe2x80x94; and D is xe2x80x94OH, xe2x80x94SH, xe2x80x94NH2, xe2x80x94NHOH, xe2x80x94SO3H, xe2x80x94OSO3H, xe2x80x94CO2H, xe2x80x94CONH2, xe2x80x94P(OH)3, xe2x80x94PO(OH)2, xe2x80x94Oxe2x80x94PO(OH)2, xe2x80x94Oxe2x80x94PO(OH)xe2x80x94Oxe2x80x94PO(OH)2, or xe2x80x94NH3+.
Another aspect of this invention relates to a pharmaceutical composition which includes a pharmaceutically effective amount of an MOAF described above and a pharmaceutically acceptable carrier. Examples of such a carrier include water, colloidal silica oxide, magnesium sterate, and cellulose.
An MOAF of this invention can be used as a photodynamic therapeutic agent to inhibit growth, including causing death, of tumor cells in a tumor site. Accordingly, this invention also relates to use of an MOAF for the manufacture of a medicament for this application.
Details of several embodiments of this invention are set forth in the accompanying description below. Other features, objects, and advantages of this invention will be apparent from the description and from the claims.
An MOAF of this invention can be synthesized by methods well known in the art. For instance, a fullerene can be first converted to a fullerene carboxylate by reacting it with a carboxylating agent such as diethyl bromomalonate. The fullerene carboxylate derivative is then oligoanilinated and, optionally, modified at the introduced oligoaniline moieties to generate ionic groups, e.g., alkylsulfonyl. Another suitable moiety, such as fullerene, can be introduced by further modifying one of the ionic groups. Each fullerene moiety can also be modified for introduction of hydrophilic groups, e.g., hydroxy, by reacting with dilute NaOH in the presence of a phase-transfer catalyst and aerated oxygen.
An effective amount of MOAF thus prepared can be formulated with a pharmaceutically acceptable carrier to form a pharmaceutical composition before being administered to a subject in need of tumor treatment. xe2x80x9cAn effective amountxe2x80x9d refers to the amount of the MOAF which is required to confer therapeutic effect on the treated subject. The interrelationship of dosages for animals and humans (based on milligrams per square meter of body surface) is described by Freireich et al., Cancer Chemother. Rep. 1966, 50, 219. Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y., 1970, 537. Effective doses will also vary, as recognized by those skilled in the art, depending on the route of administration, the excipient usage, the distance of tumor from the skin surface, the source of the irradiation, and the optional co-usage with other therapeutic treatments including use of other anti-tumor compounds. Examples of pharmaceutically acceptable carriers include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate, and DandC Yellow #10.
The pharmaceutical composition may be administered via a parenteral route, e.g., topically, intraperitoneally, and intravenously. Examples of parenteral dosage forms include an active compound dissolved in phosphate buffer solution (PBS), or admixed with any other pharmaceutically acceptable carrier. Solubilizing agents, such as cyclodextrins, or other solubilizing agents well known to those familiar with the art, can also be included in the pharmaceutical composition.
The invention also relates to the use of an MOAF of this invention or a composition containing thereof for the manufacture of a medicament for tumor. More specifically, the MOAF is administered to the tumor site and the irradiated with laser or other light sources, e.g., fluorescence or X-ray. The irradiation can be of a wavelength of 400-1000 nm and an energy intensity of 10-300 J/cm2, and the irradiation time can be 10-200 minutes. Upon irradiation, superoxide radicals are generated, which in turn attack and inhibit the growth of tumor cells.
An in vitro inhibition assay can be used to preliminarily evaluate an MOAF""s ability to inhibit the growth of tumor cells. For example, an MOAF solution can be added to a pre-incubated cell suspension. Subsequently, the cell suspension is irradiated with fluorescence light, followed by further incubation. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide solution is then added to the cell suspension to react with mitochrondrial dehydrogenase to form formazon, which is extracted with dimethyl sulfoxide (DMSO). The DMSO extract solution is immediately used for optical measurement to determine the quantity of the formazon, which correlates with the quantity of dehydrogenase or the relative number of the living cells.
The MOAFs of this invention, which have been preliminarily evaluated, can be further screened for their efficacy by an in vivo inhibition assay using tumor-bearing mice. For example, each tumor-bearing mouse can be first administered with an MOAF to be tested in PBS close to the tumor site. The mouse is then kept in the dark while the MOAF is circulated to the tumor site. The tumor site is exposed by removing the hair on and around it and then irradiated with a laser beam or other light source. The growth of the tumor in the mouse is then examined at different time intervals. The inhibitory effect is evaluated by measuring the mouse""s body weights and tumor volumes. After the mouse is sacrificed, the body weight and various organ weights are also measured, and blood samples are withdrawn for biochemistry and hematology analyses. All such data can be used to evaluate the efficacy of the MOAF to treat tumor.
Without further elaboration, it is believed that one skilled in the art, based on the description herein, can utilize the present invention to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety. The following specific examples, which describe synthesis and biological testing of several MOAFs of this invention, are therefore to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.